Functional analysis of the human endothelial nitric oxide synthase promoter. Sp1 and GATA factors are necessary for basal transcription in endothelial cells.

The Journal of Biological Chemistry
R ZhangW C Sessa

Abstract

To gain insights into the mechanisms of endothelial nitric oxide synthase (eNOS) gene expression, we have cloned the eNOS promoter and fused it to a luciferase reporter gene to map regions of the promoter important for basal transcription in bovine aortic endothelial cells (BAEC). Transfection of BAEC with F1 luciferase (LUC) (-1600 to +22 nucleotides) yielded a 35-fold increase in promoter. Progressive deletion from -1600 to -1033 (F2 and F3 LUC) did not significantly influence eNOS promoter activity. Further deletion from -1033 to -779 (F4 LUC) resulted in an approximate 40% reduction in basal promoter activity, and still further deletion from -779 to -494 (F5 LUC) did not markedly influence activity. Deletion from -494 to -166 (F6 LUC) reduced eNOS promoter activity by 40-50%. Specific mutation of the consensus GATA site (-230) in the F3 LUC construct reduced luciferase activity (by 25-30%). Gel shift analysis and antibody depletion using BAEC nuclear extracts demonstrated in vitro binding of GATA-2 to the oligonucleotide sequence containing the -230 GATA site. Next, we mutated the Sp1 site (-103) in the F3 and F6 LUC constructs and in the F3 GATA mutant construct. Expression of these Sp1 mutants in BAEC resulted in a 85-90%...Continue Reading

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